The Embden-Meyerhof (EM) or Entner-Doudoroff (ED) pathways of sugar degradation were analyzed in representative species of the hyperthermophilic archaeal genera Thermococcus, Desulfurococcus, Thermoproteus, and Sulfolobus, and in the hyperthermophilic (eu)bacterial genus Thermotoga. The analyses included (1) determination of 13C-labeling patterns by 1H- and 13C-NMR spectroscopy of fermentation products derived from pyruvate after fermentation of specifically 13C-labeled glucose by cell suspensions, (2) identification of intermediates of sugar degradation after conversion of 14C-labeled glucose by cell extracts, and (3) measurements of enzyme activities in cell extracts. Thermococcus celer and Thermococcus litoralis fermented 13C-glucose to acetate and alanine via a modified EM pathway (100%). This modification involves ADP-dependent hexokinase, 6-phosphofructokinase, and glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAP:FdOR). Desulfurococcus amylolyticus fermented 13C-glucose to acetate via a modified EM pathway in which GAP:FdOR replaces GAP-DH/phosphoglycerate kinase. Thermoproteus tenax fermented 13C-glucose to low amounts of acetate and alanine via simultaneous operation of the EM pathway (85%) and the ED pathway (15%). Aerobic Sulfolobus acidocaldarius fermented 13C-labeled glucose to low amounts of acetate and alanine exclusively via the ED pathway. The anaerobic (eu)bacterium Thermotoga maritima fermented 13C-glucose to acetate and lactate via the EM pathway (85%) and the ED pathway (15%). Cell extracts contained glucose-6-phosphate dehydrogenase and 2-keto-3-deoxy-6-phosphogluconate aldolase, key enzymes of the conventional phosphorylated ED pathway, and, as reported previously, all enzymes of the conventional EM pathway. In conclusion, glucose was degraded by hyperthermophilic archaea to pyruvate either via modified EM pathways with different types of hexose kinases and GAP-oxidizing enzymes, by the nonphosphorylated ED pathway, or by a combination of both pathways. In contrast, glucose catabolism in the hyperthermophilic (eu)bacterium Thermotoga involves the conventional forms of the EM and ED pathways. The data are in accordance with various previous reports.